Glycoprotein hormone receptors (GhRs) are a family of receptors for glycoprotein hormones secreted by the anterior pituitary. These hormones include the gonadotropins, such as luteinizing hormone (LH; lutropin), and follicle stimulating hormone (FSH; fillitropin), as well as thyroid stimulating hormone (TSH; thyrotropin), and choriogonadotropin (CG), which is produced by the placenta. Each hormone selectively binds to a member of the GhR family--LH and human CG (hCG) bind to the LH receptor (LHR), FSH binds to the FSH receptor (FSHR), and TSH binds to the TSH receptor (TSHR). The biological consequences of ligand binding to a GhR, typically mediated by an increase in the intracellular concentration of cAMP, result in steroid synthesis and secretion, as well as target cell activation and differentiation.
Situations exists where it would be desirable to efficiently express only the ligand binding portion of a selected GhR. However, past attempts to achieve such expression have been unreproducible or have resulted at best in inefficient and/or unpredictable levels of expression (Xie, et al., 1990; Tsai-Morris, et al., 1990). For example, although full length human FSH, LH and TSH receptors have been successfully expressed using the baculovirus expression system, expression of the extracellular region of both porcine LH receptor and human TSH receptor resulted in the trapping of this domain inside the host cells (Pajot-Augy, et al., 1995; Seetharamaith, et al., 1994; Chazenbalk and Rapoport, 1995). While ligand binding could be observed, the vast majority of the proteins were in a denatured, inactive and unprocessed form.
In one recent study (Bozon, et al., 1995), moderate amounts (100,000 sites/cell) of porcine LH receptor extracellular region were secreted into the media if a low expression promoter/signal peptide combination was used. However, because high expression levels invariably led to intracellular protein aggregation, it was concluded that only a moderate level of expression is compatible with production of bioactive ectodomain of the receptor. The authors (Bozon, et al., 1995) proposed to perform immunoaffinity purification of the recombinant proteins and to refold aggregated receptors using guanidine HCl in the presence of cystine and cysteine, as was done for the TSH receptor (Bobovnikova, et al., 1997).
In E. Coli, expression of the extracellular region of rat LH receptor also led to the production of self-associated protein aggregates in the inclusion bodies (Chen and Bahl, 1993). While the proteins could be refolded, the re-folding process was inefficient and difficult to do consistently, perhaps because proteins derived from prokaryotic cells lack carbohydrate side chains that are necessary to facilitate proper protein folding.
The present invention provides a convenient method, not encumbered by the above-described difficulties, for efficiently and reliably expressing such "soluble glycoprotein hormone receptors" (sGhRs). The method may also be applied to the expression of the extracellular ligand binding domains of other 7-transmembrane domain G-protein-coupled receptors having the substantial majority of their ligand binding activity in the extracellular portion of the receptor. Summary of the Invention In one aspect, the present invention includes a chimeric nucleic acid molecule formed of a 5' end segment that encodes an extracellular ligand binding region (ELBR) of a 7-transmembrane domain G-protein-coupled receptor polypeptide, a 3' end segment that encodes a membrane anchor polypeptide, and interposed between the 5' end segment and 3' end segment, a protease recognition site.
In one embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a calcitonin receptor. In a related embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a calcitonin-gene related peptide (CGRP) receptor. In another embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a glucagon receptor. In a related embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a glucagon-like peptide 1 (GLP-1) receptor. In still another embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a metabotropic glutamate receptor. In another embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a parathyroid hormone (PTH) receptor. In yet another embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a vasoactive intestinal peptide (VIP) receptor. In still another embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a secretin receptor. In an additional embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a growth hormone releasing factor (GRF) receptor.
In a general embodiment, the protease recognition site is a thrombin recognition site. In another general embodiment, the 3' end segment encodes the transmembrane portion of a CD8 molecule.
In a preferred general embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a glycoprotein hormone receptor. In another preferred general embodiment, the 3' and 5' end segments are heterologous with respect to one another.
In one embodiment, the 5' end segment encodes a human luteinizing hormone receptor binding protein (LBP). In another embodiment, the 5' end segment encodes a human follicle stimulating hormone receptor binding protein (FBP). In still another embodiment, the 5' end segment encodes a human thyroid stimulating hormone receptor binding protein (TBP).
Also included in the invention is an expression vector comprising (a) a chimeric nucleic acid molecule such as described above, and (b) regulatory sequences effective to express an open reading frame of the chimeric nucleic acid molecule in a host cell.
In another aspect, the invention includes a method of recombinantly producing a fusion polypeptide encoded by the chimeric nucleic acid molecule described above. The method includes the steps of (i) introducing into suitable host cells, the above-described expression vector, and (ii) culturing the host cells under conditions resulting in the expression of the fusion polypeptide. In a general embodiment, the method further includes incubating the host cells in the presence of a protease which recognizes the protease recognition site, to release the first segment of the chimeric polypeptide.
Also included in the invention are host cells transfected with an expression vector such as described above.
In another aspect, the invention includes a chimeric polypeptide formed of a first segment that contains an extracellular ligand binding region (ELBR) of a 7-transmembrane domain G-protein-coupled receptor polypeptide, a second segment that contains a membrane anchor polypeptide, and interposed between the first and second segments, a protease recognition site. In specific embodiments, the segments can include the corresponding polypeptide entities encoded by the above-described chimeric nucleic acid molecules. In a preferred embodiment, the first and second segments are heterologous with respect to one another.
The 7-transmembrane domain G-protein-coupled receptor polypeptide may be, for example, a calcitonin receptor, a calcitonin-gene related peptide (CGRP) receptor, a glucagon receptor, a glucagon-like peptide 1 (GLP-1) receptor, a metabotropic glutamate receptor, a parathyroid hormone (PTH) receptor, a vasoactive intestinal peptide (VIP) receptor, a secretin receptor, or a growth hormone releasing factor (GRF) receptor. In a preferred general embodiment, the 7-transmembrane domain G-protein-coupled receptor polypeptide is a glycoprotein hormone receptor.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.